PROJECT SUMMARY Large scale manipulation of neural ensemble activity at cellular resolution and millisecond precision is a major technological goal of the BRAIN initiative. Although optogenetics affords cell-type specificity, combining it with extremely high spatiotemporal resolution in functionally characterized neurons will dramatically improve the level of control and the interpretability of optogenetic experiments. Multiphoton optogenetics theoretically provides such high resolution, but suffers several critical obstacles that strongly compromise its effective resolution, speed, and scale. Here we propose to make the critical technical advances that overcome these obstacles. We aim to develop a system that achieves multiphoton holographic control of large ensembles of neurons with cellular level resolution and millisecond precision in a large volume of brain tissue, and deep into the brain. By bringing together an interdisciplinary team combining expertise in non-linear optics, neurophysiology, ion channel engineering and computational imaging, we aim to optimize this potentially breakthrough technology for broad use in the neuroscience community, and validate it in five different model organisms.